Neonatal hyperbilirubinemia, defined as a total serum bilirubin level above 5 mg/dL (86 micromolar), is a frequently encountered clinical problem. Although up to sixty percent of term newborns have clinical jaundice in the first week of life, few have significant underlying disease. High bilirubin levels that are prolonged may lead to a variety of central nervous system abnormalities, developmental delays, retardation, and other serious health impairments and even death.
Clinical jaundice typically results from the presence of high concentrations of unconjugated bilirubin pigment in the skin and mucus membranes. FIG. 2 shows the biochemical pathways for metabolizing and excreting bilirubin, as known to those of skill in the art. Depending on the underlying etiology, this condition can present throughout the neonatal period. Bilirubin is the final product of heme degradation. At physiologic pH, bilirubin is insoluble in plasma and requires protein binding with albumin. After conjugation in the liver, it is excreted in bile. Newborns produce bilirubin at a rate of approximately 6 to 8 mg per kg per day. This is more than twice the production rate in adults, primarily because of relative polycythemia and increased red blood cell turnover in neonates. The rate of bilirubin production in mg/kg/24 h typically declines to the adult rate of 3.8±0.6 mg/kg/24 h within 10 to 14 days after birth.
Although glucuronidation is one of the most important routes of biotransformation, the broad and overlapping substrate specificity of the hepatic uridine diphosphate glucuronosyltransferases UDP-glucuronosyltransferases (UGTs) that catalyze glucuronidation remains poorly understood. The two main reasons for this situation are the lack of isolated individual UGT isozymes and the lack of assay methods suitable for detecting glucuronidation of diverse compounds. The UDP-glucuronosyltransferases are a family of enzymes that catalyze the glucuronidation of endogenous compounds (such as bilirubin) and xenobiotic compounds (such as drugs), generating products that are more hydrophilic and thus more readily excreted in bile or urine. UGTs are 50-60 kDa integral membrane proteins with the major portion of the protein, including the catalytic domain, located in the lumen of the endoplasmic reticulum and a C-terminal anchoring region spanning the ER membrane. Two UGT families—UGT1 and UGT2—have been identified in humans. Although the members of these families are less than 50% identical in primary amino acid sequence, they exhibit significant overlap in substrate specificity.
The members of the UGT1 family that are expressed in human liver, where the majority of xenobiotic metabolism takes place, include UGT 1A1, 1A3, 1A4, 1A6, and 1A9. Although the UGT2 family has not been studied as extensively, it is known that UGT 2B4, 2B7, 2B10, 2B15 and 2B17 are expressed in the liver. Mutations in UGTs are known to have deleterious effects, including clinical hyperbilirubinemia.
Organic-anion transporter proteins (OATP) are cell membrane proteins that accomplish the transfer of organic compounds into and out of the liver cells so that UGT and other enzymes can metabolize the compounds. OATPs are, like UGTs, are developmentally sensitive to gestational age and other factors. OATP 1B1, 1B3, and 2B1 have been studied most to-date. Both OATPs and UGTs are involved in the metabolism and excretion of bilirubin.
Infant jaundice, or hyperbilirubinemia, is a significant clinical problem, occurring in about sixty percent of full-term and near-term infants. The syndrome is the direct result of increased bilirubin levels in the infant body. The organic-anion transport and glucuronidation metabolic pathways are not fully developed in neonates, particularly in premature neonates. Nevertheless, some near-term or term neonates, are subject to perinatal jaundice as well. Often, such infants are dismissed from hospital with moderate bilirubin levels, only to return requiring readmission to hospital several days later with markedly increased bilirubin and jaundice. High bilirubin levels that are prolonged may lead to a variety of central nervous system abnormalities, developmental delays, retardation, and other serious health impairments and even death. Apart from the morbidity associated with neonatal hyperbilirubinemia, there is significant financial loss and waste of health services resources connected with the hyperbilirubinemic readmissions to hospital.
Bilirubin is a bile pigment which is a metabolic product of heme formed from the degradation of hemoglobin in erythrocytes by reticuloendothelial cells. It can also be formed by the breakdown of other heme-containing proteins such as cytochromes. The most typical biological form of bilirubin is bilirubin IXa.
Bilirubin IXa normally circulates in the plasma of the bloodstream in several forms. One form has been designated as “unconjugated” or “indirect” bilirubin. The unconjugated bilirubin may complex to serum albumin and, as such, is designated as “bound” unconjugated bilirubin, or it may exist in a non-albumin bound or free form and, as such, is designated as “unbound” unconjugated bilirubin. Both bound and unbound unconjugated bilirubin are normally transported to the liver, wherein they are taken up by the liver cells and converted to a polar conjugate form. The conversion typically involves the transfer of glucuronic acid molecules, catalyzed by the enzyme hepatic glucuronyl transferase, to the unconjugated bilirubin. This converted bilirubin is designated in the art as “conjugated” or “direct” bilirubin. Some of the conjugated bilirubin may leak back into the bloodstream. Conjugated bilirubin in the bloodstream, like unconjugated bilirubin, can bind to albumin, although the unconjugated form seems to bind to albumin more tightly. Thus, bilirubin in the blood exists in four forms: (1) bound conjugated bilirubin, (2) unbound conjugated bilirubin, (3) bound unconjugated bilirubin and (4) unbound unconjugated bilirubin. To summarize, (1) and (2) together are known as “direct” bilirubin, while (3) and (4) together are termed “indirect” bilirubin. These four fractions generally comprise the serum or plasma bilirubin concentration. However, a fifth component, delta bilirubin, has been described which is a bilirubin covalently linked to albumin. It is typically a very small fraction of the total bilirubin.
Unconjugated but not conjugated bilirubin can poison many vital cell functions, and a variety of experimental and clinical evidence suggests that unbound unconjugated bilirubin is a potential neurotoxin. Specifically, since it is not restricted by albumin binding, unbound unconjugated bilirubin can act as a neurotoxin because of its ability to migrate from the vascular space into the nervous system where it can complex with nervous tissue causing irreversible damage. Typically, unbound unconjugated bilirubin comprises less than 0.05% of the fraction of total bilirubin in the blood and is therefore difficult to measure. Disease states resulting in elevated levels of serum bilirubin may raise either “conjugated” or “unconjugated” levels of bilirubin or both forms simultaneously. However, only elevated unconjugated and unbound forms predispose a patient to neurological bilirubin toxicity.
Newborn infants suffering from high levels of unconjugated bilirubin (i.e., hyperbilirubinemia) become jaundiced after birth and are susceptible to developing kernicterus, which is an accumulation of unconjugated bilirubin in tissues of the nervous system, particularly the basal ganglia of the developing brain. This condition, also designated as bilirubin encephalopathy, may produce athetoid cerebral palsy, ocular palsy, deafness, mental retardation, and defects in fine motor control and cognitive function. Neonates afflicted with hemolysis and infants born prematurely compose the highest risk groups for bilirubin encephalopathy; however, kernicterus has also been reported in jaundiced term newborns with no clear pathological etiology for their jaundice.
Most newborns develop transient unconjugated hyperbilirubinemia in the first few days of life. There has been a recent resurgence in bilirubin encephalopathy (kernicterus) in term and near term newborns that has been attributed both to early postnatal hospital discharge and less concern about bilirubin toxicity by health care providers.
Various treatments have been suggested for both infant jaundice when these problems occur. These treatments include phototherapy and, in some cases, exchange transfusions, extracorporeal filtration systems, and drugs which induce a more efficient clearance of bilirubin from the body. None of these treatments is simple to administer. None is effective without negative side reactions, including risk of injury or death. If the jaundice is not promptly treated, serious damage to the nervous system can result, especially in infants, as the elevated amounts of bilirubin act as a neurotoxin, and the blood/brain barrier in infants is incompletely developed. Also, the foregoing treatments are administered after the fact—i.e., after the jaundice has already appeared and typically after the jaundice has become severe and prolonged.
In neonates, the visible signs of the disorder manifest themselves usually at 72 hours or later after birth, generally after the infant has left the hospital or birth center. Thus, the signs of hyperbilirubinemia typically appear when the baby is no longer under the observation of trained medical personnel. This delayed onset of neonatal hyperbilirubinemia is also at a time when many mothers have strong psychological tendencies to hope and believe that their newborn is well. This perceptual and psychological bias often leads to significant delays in the mothers' seeking care from pediatric physicians and nurses. In order to minimize the organic and neurological damage caused by the elevated bilirubin levels, therefore, it is advantageous to intervene early, preferably prior to the baby's discharge from the hospital or birthing center and before medical control over monitoring and treatment has been lost, which is often before the visible signs of jaundice appear.
One aspect of effective intervention is the accurate and timely identification of individuals at risk for developing this syndrome. Because, in order to eliminate as totally as possible the incidence of neonatal jaundice, every infant must be tested, effective prediction requires a simple, noninvasive procedure. Measurement of bilirubin in the blood per se is not a satisfactory approach because accurate prediction of a potential to develop jaundice rests on detection of increased bilirubin production, as opposed to the levels of bilirubin in the blood. Blood bilirubin levels are influenced not only by production, of course, but also by rates of excretion, and hepatic and intestinal uptake. When subjects at risk are identified, they must either be monitored for subsequent treatment or administered a treatment in advance which prevents the onset of serious jaundice.
Prior art approaches to identification of individuals at risk for developing neonatal hyperbilirubinemia have several limitations. When only one variable is measured (such as total bilirubin, as used in the Bhutani nomogram and similar decision tools), the sensitivity and specificity are not adequate to accurately define the individual infants in the population at-risk. When one variable (such as total bilirubin) is measured serially, the invasiveness and exposure of the infant to repeated phlebotomies is usually unacceptable, to the clinicians and to the parents. When more than one variable is measured, the predictive decision-support method generally is intolerant of missing values for one or more variables and fails to recommend a decision or produce a risk-score when some information is not available. When exotic laboratory variables (such as OATP or UGT genotypes, or TNF-alpha or IL-1β, IL-6, IL-8, IL-10, or other biomarkers) are measured, one or more of the following attributes of the assay methods preclude their widespread practical implementation: excessive turnaround-time in the context of relatively short time interval that is pertinent to the clinical decision regarding what preventive or management interventions, if any, are applicable; excessive testing expense; lack of ready availability of providers of the assay methods.